The present invention relates generally to the microbial production of dyestuffs and more particularly to microbial production of indigo by organisms in indole-free media.
Indigo, or indigotin, occurs as a glucoside in many plants of Asia, the East Indies, Africa, and South America, and has been used throughout history as a blue dye. Principally obtained from plants of the genera Indigofera and Isatus, indigo was used to dye blue the earliest known textiles, linen mummy wrappings dating from 2000 BC. By the middle of the 19th century, indigo had become a principal item in trade between Europe and the Orient. Prior to elucidation of the structure and the synthesis of the indigo molecule, the use of natural indigo involved protracted fermentation processes to liberate the dye for introduction into fabric in a soluble, colorless form, indican. By steeping the fabric and indican in a vat, the soluble indican was easily hydrolyzed to glucose and indoxyl. Mild oxidation, such as exposure to air, would convert the indoxyl to indigo, regenerating the pigment in the fibers of the fabric.
During the 19th century considerable effort was directed towards determining the structure of this valuable compound. The chemical structure of indigo, corresponding to the formula C.sub.16 H.sub.10 N.sub.2 O.sub.2, was announced in 1883 by Adolf von Baeyer after eighteen years of study of the dye. However, a commercially feasible manufacturing process was not developed until approximately 1887. The method, still in use throughout the world, consists of a synthesis of indoxyl by fusion of sodium phenylglycinate in a mixture of caustic soda and sodamide. All industrially successful processes also involve the final step of air oxidation of indoxyl to indigo. To date, indigo has been principally used for dying cotton or wool shades of navy blue. The compound also has potential use in processes for solar energy collection. [See British Pat. No. 1,554,192].
Pertinent to the background of the invention are prior observations of microbial production of a blue pigment. Using selective methods of cultivation, one experimenter in 1927 isolated a soil organism (Pseudomonas indoloxidans) that could decompose indole with the formation of blue crystals. The blue particles that appeared in cultures containing that bacterium were insoluble in water, alcohol, ether, xylol, and benzol, but did dissolve in strong sulfuric acid to give a blue solution which dyed silk blue. The experimenter concluded that indoxyl was probably not formed within the cells of this organism, but rather that the blue crystal formation was due to the production of an exoenzyme diffusing out from the bacterial growth. This organism could not use indole as a source of energy and could not oxidize indole to indigotin without an additional source of carbon, but could oxidize indole if given a supply of carbon. A high carbon to nitrogen ratio appeared to be most suitable to the growth of Pseudomonas indoloxidans and the production of indigotin. Further observations made by the experimenter were that indole appeared to depress the growth of the organism and that the organisms multiplied rapidly as soon as the indole had been consumed. The oxidation of indole was observed to take place only during the early stages of growth of the organism. No trace of indoxyl was found in cultures, and the indigotin was not apparently further oxidized to isatin. The experimenter also noted that two other soil organisms, Mycobacterium globerulum and Micrococcus piltonensis, could also produce small amounts of indigotin on indole agar only. [See: Grey, P. H., "Formation of Indigotin from Indole by Soil Bacteria," Roy.Soc.Proc., B, 102: 263-280 (1927)].
A single mutant culture of Schizophyllum commune fungus producing a "blue pigment" has also been described. The culture was grown on a chemically defined, synthetic medium containing glucose, (NH.sub.4).sub.2 HPO.sub.4, thiamine, KH.sub.2 PO.sub.4, K.sub.2 HPO.sub.4, and MgSO.sub.4.7H.sub.2 O. The ammonium ion was the nitrogen source. Both a red and a blue pigment were harvested from mycelial macerates. The identification of the blue pigment extracted from the macerates with chloroform was obtained by solubility tests, absorption spectroscopy, and chemical analyses. The results of these tests were all consistent with the conclusion that the blue pigment was indigo. [See: Miles, P., et al., "The Identification of Indigo as a Pigment Produced by a Mutant Culture of Schizophyllum commune," Archives of Biochemistry and Biophysics, 62: 1-5 (1956)].
In 1962, a study was performed on the biogenesis of the pigment violacein by the organism Chromobacterium violaceum, which readily converted L. tryptophan to violacein, but did not utilize this amino acid for growth. The experimenters created a novel microbiological assay, specific for L. tryptophan, in which the quantity of violacein produced was a function of the amount of L. tryptophan present in the test sample. It was observed that when L. tryptophan was incubated with lyophilized cells, indole was transiently formed and, after a forty-eight-hour incubation, a deep blue pigment was synthesized. The pigmented material was identified as indigo on the basis of its color, absorption spectra, and RF values in thin layer chromatography. The experimenters concluded that indoxyl was an intermediate of the indigo pathway in this bacterium, and found that Chromobacterium violaceum metabolized L. tryptophan to indole by the action of tryptophanase or tryptophan synthetase. This microorganism synthesized violacein not only from L. tryptophan but also from indole. When the enzymes of the violacein pathway were inactivated by rapid lyophilization, both L. tryptophan and indole were metabolized to indigo. [See: Sebek, O. and Jaeger, H., "Divergent Pathways of Indol Metabolism in Chromobacterium Violaceum," Nature, 196: 793-795 (1962)].
In a more recent report, experimenters isolated an organism from soil by the enrichment culture techniques using indole as the sole source of carbon and nitrogen. An aerobic gram positive coccus, which rapidly decomposed indole when grown in a medium containing indole, KH.sub.2 PO.sub.4, K.sub.2 HPO.sub.4, NaCl, MgSO.sub.4, water, and yeast extract, produced a blue pigment which was not released into the culture medium. It was noted that the indole in the medium was used up very rapidly and more indole was added several times during the culture period. The cells, when harvested, were very blue and decomposed indole with the consumption of eleven to thirteen atoms of oxygen per mole of the substrate. When anthranilic acid, glucose or glycerol was substituted for indole in culturing the organism, the cells showed no ability to decompose indole, indicating that the activity was inducible. When grown on indole, the microorganism decomposed indole to hydroxyindole, anthranilic acid, and catachol. A cell-free extract of this organism contained an enzyme, dihydroxyindole oxygenase, which catalyzed the conversion of dihydroxyindole to anthranilate plus CO.sub.2. The dihydroxyindole oxygenase was determined to be an inducible enzyme which appeared only when the organism was grown on indole. The pathway proposed for degradation of indole by these experimenters was: indole to indoxyl to dihydroxyindole to anthranilic acid to catachol. [Fujioka, M. and Wada, H., "The Bacterial Oxidation of Indole," Biochemica et Biophysica Acta, 158: 70-78 (1968)].
To date, none of the above organisms has been put to use in the large-scale microbial synthesis of indigo. This is likely to be due, in large part, to unfavorable economic factors involved in providing indole as a substrate or otherwise maintaining precise nutrient balances in the growth medium.
Enteric bacteria (e.g., E.coli) indigenous to the intestinal tracts of animals are capable of accumulating indole [see, e.g., Post, et al., P.N.A.S. USA, 76: 1697-1701 (1979)] by the activity of the enzyme tryptophanase produced by the tryptophanase structural gene. Tryptophanase, believed to be a catabolic enzyme, catalyzes the degradation of tryptophan, resulting in the stoichiometric production of indole, pyruvate, and ammonia. An associated enzyme, tryptophan synthetase, can also catalyze the synthesis of tryptophan, from indole glycerol phosphate, and serine. In E.coli, synthesis of tryptophanase is inducible by tryptophan. The tryptophanase structural gene tnaA of E.coli K12 has been cloned and sequenced. See, Deeley, M., et al., "Nucleotide Sequence of the Structural Gene for Tryptophanase of E.coli K12," J.Bacteriology, 147: 787-796 (1981); and Deeley, M., et al., "Transcription Initiation at the Tryptophanase Promoter of E.coli K12," J.Bacteriology, 151: 942-951 (1982). While enteric bacteria are capable of growing on simple media, they do not possess the enzymatic wherewithal to convert indole to indigo.
Of particular interest to the background of the present invention is the inventor's copending U.S. patent application Ser. No. 419,953, filed Sept. 20, 1982, entitled "Method and Materials for the Microbiological Oxidation of Aromatic Hydrocarbons," the disclosures of which are specifically incorporated by reference herein. In this copending application, the applicant describes, inter alia, a transmissible plasmid pE317 containing a DNA sequence of Pseudomonas putida origin which codes for expression in a host microorganism of enzymes participative in the oxidative degradation of naphthalene to salicylate. Most briefly put, the copending application discloses use of plasmids such as pE317 and others to transform microorganisms such as E.coli and imbue them with the capacity to produce and accumulate selected valuable intermediates ordinarily only transitorily formed in the microbial mineralization of aromatic compounds such as naphthalene. Included in the enzymes coded for by plasmid pE317 is a naphthalene dioxygenase enzyme. This enzyme catalyzes the transformation of naphthalene to cis-1,2-naphthalene dihydrodiol. Applicant and his coworkers had previously performed an exhaustive study of the oxidation of naphthalene by a multi-component enzyme system from Pseudomonas sp.NC1B 9816 [see Ensley, et al., J.Bacteriology, 149: 948-954 (1982)] and characterized the initial reaction in naphthalene oxidation as involving an enzyme system comprised of three protein components.
At present, therefore, the art has not been provided with any reliable description of efficient microbiological production of indigo. This is the case, despite knowledge of the existence of certain microorganisms having the capacity to synthesize and accumulate indole and certain other organisms having the capacity to employ indole as a substrate for indigo synthesis.